Development of the retinotectal projection in zebrafish embryos under TTX-induced neural-impulse blockade

The influence of neural activity on the morphology of retinal-axon-terminal arbors and the precision of the developing retinotectal projection in zebrafish embryos was explored. Terminal-arbor morphology and their distribution in the tectum was determined with anatomical fiber-tracing methods using the fluorescent dyes dil and diO. To allow development under activity-deprived conditions, TTX was injected into the eyes of 30-38-hr-old zebrafish embryos at concentrations that effectively blocked neural activity both in retinal ganglion cells and throughout the CNS. Much like axons with normal neural-activity patterns, activity-deprived axons from dorsal and ventral and from temporal and nasal regions in the retina terminated over retinotopically appropriate and nonoverlapping regions of the tectum. Even after ablation of 1 hemiretina at the time of axonal outgrowth, activity-deprived axons from the remaining hemiretina grew directed toward and arborized selectively within their retinotopically appropriate tectal half in the same way as would nondeprived axons. Besides being retinotopic, the area over which small populations of activity-deprived axons from neighboring ganglion cells arborize is as small as that of active axons. The size of terminal arbors of retinal ganglion cell axons was unaffected by blockade of neural activity. The mean terminal-arbor size was 27 x 18 microns for the TTX-injected and 31 x 22 microns for the control embryos. The tectal coverage of TTX-blocked and control axons was equally small, with values of 1.4% and 1.6%, respectively. These data show that a precisely organized retinotopic map in developing zebrafish forms independent of neural-impulse activity.     

Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order

The retinotectal projection is organized in a precise retinotopic manner. We find, though, that during development the growth and arborization of temporal retinal axons within the optic tectum of chick embryos is initially imprecise. Axonal targeting errors occur along the rostral-caudal and medial-lateral tectal axes, and arbors are formed at topographically inappropriate positions. Subsequent course corrections along both tectal axes and large-scale axonal remodeling lead to the retinotopic ordering of terminal arborizations characteristic of the mature projection. The trajectories and branching patterns of temporal retinal axons labeled with Dil or DiO were determined in whole mounts of retina and tectum from chicks ranging in age from embryonic day 9 to posthatching. Within the retina, labeled retinofugal axons travel in a compact bundle but do not maintain strict neighbor relations, as they course to the optic fissure. The axons enter the contralateral tectum at its rostral edge and grow caudally. Many extend well past their appropriate terminal zone within rostral tectum; a proportion of these later reverse their direction of growth. Many axons grow onto the tectum at incorrect positions along the medial-lateral tectal axis. Some correct this error in a directed manner by altering their trajectory or extending collateral branches at right angles. About 80% of the positional changes of this type are made in the direction appropriate to correct axon position, and thus are likely a response to tectal positional cues. After maturation of retinotopic order, about half of the axons that project to a mature terminal zone have made abrupt course corrections along one or both tectal axes, indicating that initially mistargeted axons can establish appropriately positioned arbors and survive. The development of temporal axons within the tectum is characterized by 3 phases: elongation, branch and arbor formation, and remodeling. After considerable rostrocaudal elongation, an axon typically develops numerous side branches and arbors, many at inappropriate locations. Most arbors are formed by side branches that develop as interstitial collaterals; few axons grow directly to their appropriate terminal zone and arborize. Aberrant arbors, and axons and axon segments that fail to form arbors in the appropriate terminal zone, are rapidly eliminated over about a 2 d period. Axon degeneration appears to play a role in this remodeling process.     

Trajectories of regenerating retinal axons in the goldfish tectum: I. A comparison of normal and regenerated axons at late regeneration stages

To visualize and compare the intratectal path of normal and regenerated retinal axons, HRP was applied to localized sites in the dorsotemporal and dorsonasal retina in normal goldfish and in goldfish at 3-12 months after optic nerve section. The anterogradely labeled axons were traced in tectal whole mounts. In normal animals the axons were confined to the appropriate ventral hemitectum. Therein they ran in very orderly routes (Stuermer and Easter: J. Neurosci. 4:1045-1051, '84) and terminated in regions retinotopic to the labeled ganglion cells in the retina. The terminal arbors of dorsotemporal axons resided in the ventrorostral tectum and those of dorsonasal axons in the ventrocaudal tectum. In regenerating animals the terminal arbors also resided at retinotopic regions, where they sometimes formed two separate clusters. In contrast to normal axons, the regenerating ones traveled in abnormal routes through the appropriate and inappropriate hemitectum. From various ectopic positions, they underwent course corrections to redirect their routes toward the retinotopic target region. In their approach toward their target sites, dorsotemporal and dorsonasal axons behaved differently in that the vast majority of dorsotemporal axons coursed over the more rostral tectum whereas dorsonasal axons progressed into the caudal tectal half. This differential behavior of regenerating dorsonasal and dorsotemporal axons was substantiated by a quantitative evaluation of axon numbers and orientations.     

Recovery of regenerating goldfish retinal ganglion cells is slowed in the absence of the topographically correct synaptic target

When the axons of goldfish retinal ganglion cells are severed the cell bodies undergo a series of changes as the axons regenerate. These changes begin to reverse when the axons start to innervate the tectum and by 3 months after the lesion the cell bodies have nearly returned to normal. When the axons projecting to the caudal tectum were severed by a mediolateral transection of the tectum, only retinal ganglion cells in the nasal portion of the contralateral retina underwent the changes normally associated with regeneration, followed by a speedy return to normal. Because the injured fibers probably did not fully retract from the tectum, these results indicated that: (1) the complete removal of the axons from the tectal milieu was not essential for initiating the cell body changes, and (2) close proximity to the target sites would speed the recovery of the cells. When the caudal portion of the tectum was ablated the retinal ganglion cells of the nasal retina remained enlarged significantly longer than after tectal transection. During the time the cells remained enlarged the electrophysiological projection onto the remaining rostral part of the tectum revealed no significant 'compression' of the visual field. Compression of the visual field onto the rostral portion of the tectum can be accelerated if the caudal tectal ablation is accompanied by an optic nerve crush. However, under this condition the recovery of ganglion cells in the nasal retina was significantly slower than the recovery of cells in the temporal retina. This may reflect an element of topographical specificity in the regulation of the recovery of the cell body from axonal injury.     

Evidence for the stability of positional markers in the goldfish tectum

Positional markers in the tectum, which are thought to guide growing axons to their target sites, have been proposed to be induced by axons, to be only transiently associated with the tectal cells, and then lost after long-term denervation periods (Schmidt: J. Comp. Neurol. 177:279-300, '78). To further investigate this concept, retinal axons were induced to regenerate into ipsilateral tecta which had been deprived of their retinal afferents for shorter (0-4 months) and longer periods (4-8 months). The paths of HRP-labeled regenerating axons of known retinal origin were traced and used as an operational test to decide whether the axons might navigate under the influence of positional markers. Two different kinds of experiments were performed: 1. The axons from a subpopulation of all ganglion cells in the retina were labeled by applying a small crystal of HRP at defined retinal regions. Independent of the denervation period of the tectum, the labeled regenerating axons traveled in abnormal but nonrandom routes. In early regeneration stages, axons exhibited signs of exploratory growth. They extended branches equipped with growth cones and filopodia into various regions of the tectum. In late regeneration stages, the axons lost these branches, exhibited U-turns and bends, and ended in terminal arbors in the retinotopic target region. These findings suggest that the axons travel under the influence of tectal positional markers and that these markers are not transient. 2. Axons from a surgically created temporal hemiretina were labeled by application of HRP to the optic nerve to test whether the temporal axons might expand into the caudal tectum in long-term-denervated tecta. The HRP-labeled axons coursed over rostral and midtectal regions. Instead of invading the caudal tectum they bent and terminated in the rostral tectal half. These results add further support for the conclusion that the path of regenerating retinal axons is governed by long-lasting positional markers.     

Effects of permeability of midtectal barriers in goldfish on compression of the visuotectal projection rostrally and regenerative escape caudally

Physiological mapping and anatomical methods were used to evaluate changes in the retinotectal projection of goldfish 16-200 days after insertion of permeable or impermeable barriers that bisected the tectum into rostral and caudal halves. The projection to rostral tectum was left intact. Barriers composed of Gelfilm or impermeable Nucleopore material induced within 2-3 months an orderly compression of the visual field representation in rostral tectum only slightly less complete than that observed in animals with caudal half-tectal ablation. In contrast, Nucleopore filter barriers with 0.1-micron or 8-micron holes did not cause significant compression. According to both mapping and autoradiographic tracing, reinnervation of tectum behind the barriers occurred among all groups within 1-2 months. Physiologically, the projection caudal to permeable barriers was typically complete and appropriate, whereas the caudal projection in fish with impermeable barriers eventually consisted of a greatly expanded representation of the extreme temporal visual field. Autoradiography, normal fiber impregnations, and the orthograde horseradish peroxidase method revealed that regeneration past the barriers involved the formation of large bundles passing vertically along the cut tectal margin and through the underlying valvula cerebelli or lateral tegmentum. The simultaneous rostral compression and caudal expansion in the visual representation formed when more impermeable barriers were used provides evidence that, in addition to the influence of position-dependent properties, axonal competition for target territory contributes to the control of the distribution of optic arbors. Further research is required to determine why reinnervation of tectum caudal to the more permeable barriers was more complete with respect to visual representation.     

Rules of order in the retinotectal fascicles of goldfish

Individual fascicles of retinal axons were labeled in the goldfish tectum with horseradish peroxidase (HRP). The contralateral retina was later processed for HRP histochemistry to mark the cells that had axons in the fascicles. Labeled cells were found in a partial half anulus in ventral hemiretina, centered on the optic disk. The distance of the partial anulus from the disk depended on which tectal fascicle had been labeled; the more rostrocentral the fascicle, the smaller was the annular radius. The angular subtense of the partial anulus with respect to the disk depended on where (along its tectal course) the fascicle had been labeled; the more rostral the label site, the longer was the angular subtense. These results were interpreted in the context of retinotectal growth, and it was inferred that the axons followed two rules: (1) grow in along the edge of the tectum and (2) exit and terminate in order, axons from temporal retina first, nasal retina last. These rules would produce a retinotopic projection in peripheral tectum, but they require that some of the terminals already in place must shift as the tectum grows.     

Changes in the topographically organized connections between the nucleus isthmi and the optic tectum after partial tectal ablation in adult goldfish

The projection of the nucleus isthmi to the ipsilateral optic tectum was examined in normal goldfish. This was compared to the projection in animals in which the entire visual field had been induced to compress onto a rostral half tectum by caudal tectal ablation. The isthmo-tectal projection was examined by making localized injections of horseradish peroxidase into the optic tecta and observing the patterns of labeled cells within the nucleus isthmi. The teleost nucleus isthmi consists of a cell sparse medulla covered by a cellular cortex, which is thick on the rostral, medial, and dorsal surfaces of the nucleus. Almost all isthmic cells projecting to the tectum were located in the area of thick cortex. In normal fish, rostral tectal injections labeled cells in the rostroventral portion of the thick cortex; injections midway in the rostrocaudal tectal axis labeled more caudodorsally located cells, and caudal tectal injections labeled cells a little further caudally in extreme dorsal cortex. The rostroventral to caudodorsal isthmic axis was therefore seen to project rostrocaudally along the tectum. This topography contrasts somewhat with the situation seen in amphibia where the rostrocaudal tectal axis receives projections from the rostrocaudal isthmic axis. In fish with half-tectal ablations, injections near the caudal edge of the half tectum (at a site that had originally been midtectal) labeled cells that had previously projected to caudal tectum. Rostral tectal injections in fish with compression of the visual field gave a normal pattern of labeled isthmic cells. The results indicate that a topographically ordered isthmo-tectal projection exists in goldfish that may be induced to compress onto a half tectum.     

Tectoreticular pathways in the turtle, Pseudemys scripta. I. Morphology of tectoreticular axons

Tectoreticular projections in turtles were examined by reconstructing from serial sections axons that were anterogradely filled with horseradish peroxidase after tectal injections. Three tectoreticular pathways each contain extensively collateralized axons. The crossed dorsal pathway (TBd) contains large and small caliber axons. After leaving the tectum, TBd axons emit collaterals into the ipsilateral profundus mesencephali rostralis and then give off a main rostral branch that bears secondary collaterals in the ipsilateral interstitial nucleus of the medial longitudinal fasciculus and the suprapeduncular nucleus. The main trunks cross the midline and descend in the predorsal bundle, generating collaterals at regular intervals. These terminate mostly in the medial half of the reticular core from the midbrain to the caudal medulla. Axons in the uncrossed intermediate pathway also emit collaterals into a midbrain reticular nucleus (profundus mesencephali caudalis) and often have a thick rostral branch. The main caudal trunks, however, remain ipsilateral and travel in a diffuse, laterally placed tract, where each emits a long series of collaterals into the lateral half of the reticular core. The uncrossed ventral pathway (TBv) contains medium and small caliber axons. TBv axons often have collaterals within the tectum and apparently lack main rostral branches. Their caudal trunks run in the tegmental neuropile below the TBi where they collateralize less exuberantly than do TBd and TBi axons. The morphology of axons in all three pathways suggests that projections from disjunct tectal loci converge at many rostrocaudal levels within the reticular formation. This point was examined explicitly in experiments in which two disjunct injections were placed in one tectal lobe. Intermediate pathway axons traced from the two loci initially formed two distinct bundles but then intermingled in the reticular formation.     

Evidence for centripetally shifting terminals on the tectum of postmetamorphic Rana pipiens

In larval frogs the retina and tectum grow in topologically dissimilar patterns: new cells are added as peripheral annuli in the retina and as caudal crescents in the tectum. Retinotopy is maintained by the continual caudalward shifting of the terminals of the optic axons. After metamorphosis the pattern of growth changes. The retina continues to add new ganglion cells peripherally, but there is no neurogenesis in the tectum. To maintain retinotopy in postmetamorphic frogs, the terminals of the optic axons must continually shift toward the central tectum. We tested the proposal of centripetally shifting axons by making punctate injections of horseradish peroxidase (HRP) in the tectum of adult Rana pipiens and observing the patterns of filled cells in the contralateral retina, as was done in the goldfish (Easter and Stuermer, '84). Punctate applications of HRP in the tectum should be taken up: 1) by fascicles, and label a partial anulus of cells, 2) by terminals, and label a cluster of cells in the corresponding retinotopic site, and 3) by the extrafascicular axonal segments, and label a band of cells connecting the partial annulus to the cluster. If the terminals have shifted centripetally, the band of cells labeled through their extrafascicular segments should have a spoke-like orientation, with the center of the retina as the hub. As the tectal site moves from rostral to caudal, this band of cells should move, pendulum-like, from temporal to nasal retina. In general, the patterns of HRP-filled retinal cells we observed were consistent with our predictions. In addition, HRP taken up by the oldest (rostral) tectal axons produced more complex patterns of filled cells that indicated that these axons had shifted both caudally before metamorphosis and centripetally after.     

Topography of the retinal projection to the superficial pretectal parvicellular nucleus of goldfish: a cobaltous-lysine study

The retinal projection to the superficial pretectal parvicellular nucleus (SPp) of goldfish was examined by filling select groups of optic axons with cobaltous-lysine. The tracer was applied intraocularly to peripheral retinal slits in some fish. In other fish, it was applied to optic axons from an intact hemiretina after one-half of the retina was ablated and the corresponding optic axons had degenerated. The results indicated that SPp is a folded structure, having a dorsal surface innervated by axons from temporal retinal ganglion cells and a ventral surface innervated by axons from nasal retinal ganglion cells. Peripheral retina innervates the anterodorsal and anteroventral edges of SPp, while central retina innervates the posterior genu. Dorsal retina innervates lateral SPp and ventral retina innervates medial SPp. Thus, although SPp is a folded nucleus, the topography of the retino-SPp projection is similar to the topography of the retinotectal projection. That is, the relative position of optic axons within SPp mirrors the retinal location of the ganglion cells that project to SPp. Retino-SPp axons occupy the center of the main optic tract before it divides into the two optic brachia. These axons are topographically arranged, with temporal retino-SPp axons being flanked on both sides by nasal retino-SPp axons. Retino-SPp axons arborize within SPp and then continue to enter the superficial tectal retino-recipient lamina. Thus, these axons innervate both SPp and the optic tectum. These findings are discussed with respect to chemospecific and morphogenetic views of visual system topography.     

Trajectories of regenerating retinal axons in the goldfish tectum: II. Exploratory branches and growth cones on axons at early regeneration stages

HRP was applied to small sites in the dorsotemporal or dorsonasal retina in fish at 10-36 days after optic nerve section. The anterogradely labeled axons were visualized in tectal whole mounts. Axons traveled through all regions of the tectum in various abnormal routes. Misrouted axons were also seen to alter their orientation and to direct their course toward their target. At all regeneration stages the majority of dorsotemporal axons coursed and achieved target-related orientations preferentially within the rostral tectal half whereas dorsonasal axons proceeded into the caudal tectum. The growing axons exhibited various morphologies. All axons in the superficial fascicle layer stratum opticum (SO) and some in the synaptic layer stratum fibrosum et griseum superficiale (SFGS) were unbranched and tipped with a leading growth cone. Other axons in the synaptic layer carried one to several growth cones at their ends and often filopodia proximal to the growth cone, or they had sprouted numerous side branches with growth cones and filopodia on the shaft and on branches. Some axons at retinotopic or ectopic sites gave rise to several long branches of several hundred microns in length, with growth cones and filopodia. From 32 days onward axons ending in terminal arbors at retinotopic sites became apparent. Thus, numerous axons at early regeneration stages go through a phase of exploratory growth on their way toward their target sites.     

Mode of growth of retinal axons within the tectum of Xenopus tadpoles, and implications in the ordered neuronal connection between the retina and the tectum

Retinal axons of Xenopus tadpoles at various stages of larval development were filled with horseradish peroxidase (HRP), and their trajectories and the patterns of branching within the tectum were analyzed in wholemount preparations. To clarify temporal and spatial modes of growth of retinal axons during larval development, special attention was directed to labeling a restricted regional population of retinal axons with HRP, following reported procedures (H. Fujisawa, K. Watanabe, N. Tani, and Y. Ibata, Brain Res. 206:9-20, 1981; 206:21-26, 1981; H. Fujisawa, Dev. Growth Differ 26:545-553, 1984). In developing tadpoles, individual retinal axons arrived at the tectum, without clear sprouting. Axonal sprouting first began when growing tips of each retinal axon had arrived at the vicinity of its site of normal innervation within the tectum. Thus, the terminals of the newly added retinal axons were retinotopically aligned within the tectum. The retinotopic alignment of the terminals may be due to an active choice of topographically appropriate tectal regions by growth cones of individual retinal axons. The stereotyped alignment of the newly added retinal axons was followed by widespread axonal branching and preferential selection of those branches. Each retinal axon was sequentially bifurcated within the tectum, and old branches that had inevitably been left at ectopic parts of the tectum (owing to tectal growth) were retracted or degenerated in the following larval development. The above mode of axonal growth provides an adequate explanation of cellular mechanisms of terminal shifting of retinal axons within the tectum during development of retinotectal projection. Selection of appropriate branches may also lead to a reduction in the size of terminal arborization of retinal axons, resulting in a refinement in targeting.     

Development of the transient ipsilateral retinotectal projection in the chick embryo: A numerical flourescence-microscopic analysis

The ipsilateral retinotectal projection in the developing chick was examined by using rhodamine-B-isothiocyanate (RITC)as an anterograde and retrograde vital marker for the retinal ganglion cells and their axons. Staining of the entire retina following intravitreal RITC injection between incubation days 3 and 16 revealed a small number of anterogradely labeled fibers in the optic tract and the anterior half of the optic tectum ipsilateral to the injection site. The total number of ipsilaterally projecting fibers was estimated to be about 2,000 on developmental day 9. The ipsilateral projection totally disappeared after day 15. The arrangement of fibers within the ipsilateral projection was examined by local anterograde RITC staining of localized retinal regions between days 9 and 10. The projection was retinotopically organized along the dorsoventral axis such that fibers of dorsal retinal origin projected on the ventral tectal half, whereas fibers of ventral retinal orgin projected on the dorsal tectal half. The localization of ipsilaterally projecting ganglion cell bodies was examined by retrograde RITC staining during days 9 and 15. Ganglion cells of all four quadrants of the central retina contributed to the production of the ipsilateral projection. The ipsilaterally growing retinotectal fibers did not represent collaterals of contralaterally projecting retinotectal axons. We assume that the tendency of early growing retinotectal axons to grow straight, as well as the ability of axonal growth cones to “sample” the environment, lead to a crossing of axons to the contralateral side. Ipsilateral projections would therefore represent “pathfinding errors.” Explanations for the elimination of the ipsilateral retinotectal projection are discussed.     

Reorganization of retinotectal projection following surgical operations on the optic tectum in goldfish

The pattern of neural reconnection between the retina and surgically operated tectum was studied in juvenile goldfish (8–11 cm long) with electrophysiological methods. The results confirm that the remaining rostral half-tectum reacquires a complete visual projection from the whole retina about 90 days after excision of the caudal half. The same reorganization of visual projection from the whole retina onto the rostral half-tectum was found to occur in the presence of the caudal half of the tectum, if the two halves were separated by a transverse surgical incision down to the level of the optic ventricle regardless of whether the contralateral optic nerve was left intact or crushed to regenerate. The reorganization of retinotectal projection was also found to occur biaxially along the mediolateral as well as the rostrocaudal axis of the tectum following excision of a caudomedial sector of the tectum. It is suggested that the reorganization of retinotectal projection is due to synaptic respecifications of individual tectal neurons in correct retinotopic order, and that the cellular discontinuity between the rostral and the caudal parts of the tectum is sufficient to induce the orderly synaptic respecifications in juvenile goldfish.     

Tests for neuroplasticity in the anuran retinotectal system

Mapping of retinotectal projections in the tree frog Hyla regilla was carried out by both behavioral and electrophysiological recording techniques following tectal ablations, with and without optic nerve regeneration. Scotomata produced by unilateral and bilateral half tectum ablations and by unilateral rectangular midtectal excisions were found to remain essentially unaltered in all cases through recovery periods up to 334 days. Similarly, electrophysiological mapping of the rostral half tectum separated by Gelfilm implants from the caudal tectum for up to 191 days yielded a normal rostral visual field. The stability of the retinotectal projection in adult Hylidae observed in these experiments contrasts with the plastic readjustments obtained in young goldfish in which the retinotectal system is still probably growing and presumably capable of field regulation. The results are taken to support the original explanatory model for developmental patterning of retinotectal connections in terms of selective cytochemical affinities between retinal and tectal neurons.     

Spatial organization of the retinal projection to the avian lentiform nucleus of the mesencephalon

Utilizing the horseradish peroxidase retrograde tracing technique and the 2-deoxy-D-glucose metabolic mapping technique, we have demonstrated in chickens the distribution of retinal ganglion cells that project to the lentiform nucleus of the mesencephalon (LM) and the retinotopic organization of the projection in the LM. Retinal ganglion cells labeled after a nearly complete injection into the LM were found in the four quadrants, distributed in a wide horizontal belt lying along both sides of the retinal equator and stretching from the temporal to the nasal retina. The HRP-labeled cells, which appeared round or oval, ranged from 25 to 840 micron 2 in size with most in the smaller size range. Results of partial HRP injections into the LM and metabolic mapping patterns in the LM produced by stimulation of half the retina with horizontal visual motion suggest that there is an orderly mapping of the retina onto the LM. The inferior temporal quadrant projects to the rostrodorsal LM; the inferior nasal quadrant projects to the caudodorsal LM. The superior temporal quadrant projects to the middle and ventral LM, extending from the rostral to the caudal pole, whereas the superior nasal quadrant projects to a small zone in the caudal LM. The mapping of the retinal quadrants in the LM is remarkably similar to that reported in the optic tectum of birds. We suggest that a common embryological anlage with the optic tectum and the arrangement of retinal axons in the optic tract are important factors in establishing the retinotopic organization of the LM.     

Pathfinding and target selection of goldfish retinal axons regenerating under TTX-induced impulse blockade

To define the extent to which impulse blockade interferes with the morphological changes of regenerating retinal axons during their growth through the tectum, axons were deprived of activity by repeated intraocular injections of TTX. At intervals between 24 and 189 days after optic nerve section (ONS), a defined group of TTX-silenced axons and of axons with normal activity (controls) were labeled by applications of HRP to the ventro- or dorsotemporal retina. The trajectories of these labeled axons were traced in DAB processed tectal wholemounts. As in controls, TTX-blocked axons went through a phase of exploratory growth at early regeneration stages (24 to 80 days after ONS). Coursing in abnormal routes, the axons initially distributed their growing endings widely over the tectum. Axons with and without activity extended side branches with growth cones and filopodia over all regions of the tectum. These ramifications were of similar dimensions for the TTX-blocked and control axons. Despite abnormal routes and branching over inappropriate territories, axons showed a preference for the rostral tectum. At late regeneration stages (120-189 days after ONS), axons had lost their side branches and their growth cones. Their preterminal segments exhibited striking bends, suggesting that they had undergone course corrections to achieve access to the retinotopic target. Axonal processes had disappeared from the caudal tectum, and the preferential accumulation of axons over the rostral tectum had increased. The majority of the TTX-blocked and control axons ended in terminal arbors at retinotopic regions. The labeled arbors of the TTX-group were no larger than those of the control group. The arbors of each group lay close together in a continuous cluster in the TTX-group as well as in two-thirds of the control group. In the other one-third of the control group, however, terminal arbors were aggregated into separate patches. The clusters of the TTX-blocked axons covered between 2.2 and 3.9% (mean 2.95%) of the tectal surface and the clusters and/or patches of active axons between 1.9 and 3.4% (mean 2.7%). Thus the terminal arbor clusters of the TTX-silenced axons were not significantly larger than those of the active axons. These data show that retinal ganglion cell impulse activity is required for neither the extension of side branches in the early exploratory phase of regeneration nor for the withdrawal of these branches nor for the establishment of target-directed routes and the deployment of normal-size terminal arbors at retinotopic loci.(ABSTRACT TRUNCATED AT 400 WORDS)     

Map formation in the developing Xenopus retinotectal system: an examination of ganglion cell terminal arborizations

Single axonal arbors of retinal ganglion cells have been stained by injecting cobalt extracellularly into the retinae of Xenopus embryos and tadpoles. The axonal endings of the earliest retinal axons to arrive in the midbrain were usually simple in appearance, often ended in growth cones, and terminated in tectal regions appropriate to their location in the eye. Thus, a topographic projection exists very early in the development (stages 37 to 39) of the projection, before the elaboration of complex axonal arbors. Retinal axons began acquiring more mature features, exemplified by the elaboration of terminal arbors, by stage 39. The arbors of most ganglion cells were elongated in the rostral-to-caudal dimension during early larval life (stages 40 to 45) and covered a large portion of tectal neuropil. During mid-larval stages (stages 46 to 50), arbors covered a relatively smaller proportion of the tectal neuropil. A quantitative analysis of this change suggests that the apparent decrease in size of the arbors, with respect to the tectum, is due to rapid growth of tectal neuropil and not due to retraction of an initially diffuse arbor. Thus, the refinement in targeting of axonal arbors during development is a phenomenon distinct from that seen during regeneration.     

Selective retinal reinnervation of a surgically created tectal island in goldfish. II. Electron microscopic analysis

In the preceding study (Edwards et al., '85), we showed that regenerating optic axons reestablish a topographically restricted projection to a caudal tectal island created by surgical removal of a 1-mm-wide strip of caudal tectum in goldfish. In the present ultrastructural study, we evaluated the dependence of this axonal outgrowth on the presence of tectal target tissue caudal to the gap. Axon counts in the lesion zone were compared between cases with complete caudal tectal ablation and cases with ablation sparing a caudal tectal island (with and without optic nerve crush). During the postoperative interval of 20-50 days (early period), up to about 1,000 unmyelinated axons with features characteristic of optic axons were present in numerous small subpial bundles in both preparations. In the subsequent interval of 50-110 days (middle period), less than 200 axons were counted caudal to simple half-tecta, whereas 4,000-14,000 myelinated and unmyelinated axons were present in a few large bundles which crossed the lesion zone of tectal island cases. In this period, optic terminals could be demonstrated in the tectal island using the anterograde horseradish peroxidase method. At 170-300 days after surgery (late period), bridging bundles contained between 2,000 and 6,000 largely myelinated axons. We conclude that caudal tectal tissue is not necessary for the initial outgrowth of a small number of axons beyond a rostral half-tectum. The target is essential, however, for the maintenance of these axon fascicles and for the subsequent massive outgrowth of axons to the island. The contributions of glial guidance, diffuse exploratory outgrowth, and target-produced trophic factors to the formation of an initially exuberant projection to the island are discussed. A process of selective axon collateral withdrawal is proposed to account for the decrease in axon numbers within bridging bundles in the late period and for the late restriction in the retinal origin of the island projection indicated by results in the preceding study (Edwards et al., '85).